Yes / The integrin family of cell surface receptors integrates cell-extracellular matrix interactions with the cell cytoskeleton and signalling across the cell membrane, resulting in an important role in cell adhesion, mobility and migration, proliferation, and survival. Changes in the number and identity of integrin receptors are common in cancer cells resulting in alteration of the ability of malignant cells to interact with the extracellular matrix, and promoting migration as well as facilitating survival outside the tumour normal environment. Beta3 integrins are potentially involved in every step of the metastatic process and expression of both alphaIIbbeta3 and alphaVbeta3 is correlated with metastatic ability of tumour cells. The recognition of the RGD binding motif common to the disintegrins and natural integrin ligands such as fibrinogen allowed the development of small molecule beta3 integrin antagonists, progressing from linear peptides containing the RGD sequence to cyclic peptides with well-defined conformation, and hence to small molecule peptidomimetics with improved pharmacological properties. In this review, we summarize the role of the beta3-subfamily of integrins when expressed in normal and tumour tissue, the development of small-molecule antagonists of beta3 integrins and their potential anti-cancer applications / EPSRC, Yorkshire Cancer Research

Despite recent advances in early detection, in 2017 prostate cancer is estimated to claim over 26,000 lives in the United States alone. Prostate cancer related morbidity and mortality is a result of secondary skeletal metastasis. Therefore, better understanding of the underlying molecular mechanisms of prostate tumor cell migration and subsequent metastasis is vital for improved clinical outcomes. Interestingly, integrin α6, a laminin receptor, is highly expressed in a number of aggressive tumor types including prostate and is associated with increased metastasis and reduced patient survival. Preliminary studies by our group found that α6 integrin undergoes a post-translational modification mediated by the serine protease, uPA and its receptor, uPAR, leading to the cleavage of α6 integrin and production of the tumor specific structural variant integrin α6p. Cleavage of this laminin receptor and production of α6p variant gives rise to an aggressive phenotype, markedly increasing tumor cell migration and invasion. Thus, the work conducted here sought to identify the function and efficacy of these prominent proteins in various aspects of tumor cell migration as well as the factors regulating α6 integrin cleavage. Interestingly, utilization of a co-culture system of prostate tumor cells and macrophages found that a direct and indirect interaction between the two cell populations influenced α6 integrin cleavage. Specifically, prostate tumor cell interactions with macrophages, a known immune cell population that is highly observed in a number of primary tumors, resulted in increased protein levels of uPAR on the surface of prostate tumor cells that led to a significant production of α6p and subsequently increased invasion. Additionally, key downstream effectors of integrin signaling, including FAK, ILK, and actin, were found to regulate production of the tumor specific variant integrin α6p. Depletion of FAK, ILK, or actin, resulted in a significant increase in uPAR protein expression and subsequent α6 integrin cleavage, a regulatory event previously not known of these integrin signaling effector molecules. In addition, silencing of another prominently expressed laminin receptor, integrin α3β1, led to a significant increase in the cohesive collective phenotype exhibited by aggressive prostate tumor cells that was found to be facilitated by α6 integrin cleavage. Depletion of integrin α3β1 resulted in increased surface uPAR expression and increased lateral association with α6 integrin, which resulted in a striking increase in α6p production, a novel finding showing the regulation of one laminin receptor is dependent on the expression of another. Furthermore, the expression of α6 integrin as well as uPAR, was found to be highly expressed in aggressive pancreatic tumor cells. This expression pattern was found to significantly increase in response to the development of drug resistance and increased invasive potential. This finding showed a never before seen efficacy of integrin and uPAR expression in dictating acquired drug resistance in pancreatic tumor cells and demonstrates their potential use as prognostic biomarkers for acquired chemotherapy resistance. Taken together, the work conducted here illustrates the utility in further understanding the role of integrins and their regulation in mediating tumor cell migration and subsequent metastasis in the progression of aggressive epithelial cancers.

Yes / The Arg-Gly-Asp (RGD)-binding family of integrin receptors, and notably the β3 subfamily, are key to multiple physiological processes involved in tissue development, cancer proliferation, and metastatic dissemination. While there is compelling preclinical evidence that both αvβ3 and αIIbβ3 are important anticancer targets, most integrin antagonists developed to target the β3 integrins are highly selective for αvβ3 or αIIbβ3. We report the design, synthesis, and biological evaluation of a new structural class of ligand-mimetic β3 integrin antagonist. These new antagonists combine a high activity against αvβ3 with a moderate affinity for αIIbβ3, providing the first evidence for a new approach to integrin targeting in cancer. / This work was supported by the EPSRC (RCUK Academic Fellowship and Grant EP/H002626/1 to H.M.S.) and Prostate Cancer UK (Pilot Grant PA10-01). F.O.F.O.A-S.. was funded by the Public Authority for Applied Education and Training, Kuwait (PhD studentship).

No description available.

Integrins are major adhesion receptors for the extracellular matrix (ECM). This thesis focuses on the motifs and interactions within integrin cytoplasmic tails during integrin-mediated cell adhesion and spreading. The present study investigated the significance of the skelemin-αIIbβ3 interaction using Chinese Hamster Ovary (CHO) cells expressing wild-type or mutant αIIbβ3 receptors defective in skelemin binding. Most mutant cells displayed unimpaired adhesive capacity and spreading on immobilized fibrinogen at the early stages of cell spreading. In addition, they formed normal focal adhesions and stress fibers with no indication of impaired cell spreading. K716A and H722A mutant cells exhibited the greatest cell spreading, which was associated with enhanced p-Src activation. The K716 residue appeared to be the most important for skelemin binding in previous in vitro studies. Here, the protrusions of the leading edge of K716A cells showed strong colocalization of talin with αIIbβ3 which was associated with a loss in skelemin binding. These data suggest that the binding of skelemin to αIIbβ3 is not essential for normal cell spreading, but may act to exert contractile forces on cell spreading and coordinate the binding of talin to the membrane proximal region of integrin tails. The functional mode of peptides corresponding to the central motifs of the αIIb and αV tail, KRNRPPLEED (αIIb peptide) and KRVRPPQEEQ (αV peptide) was also investigated. Both peptides inhibited Mn2+-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing CHO cells to immobilized fibrinogen. Breast cancer progression has been linked to tumor cell interaction with ECM. Our αIIb and αV peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV integrin ECM ligand vitronectin. Replacement of RPP with AAA significantly attenuated the inhibitory activity of the αIIb peptide. β-tubulin was identified as a potential αIIb peptide-binding partner, suggesting that microtubule cytoskeleton may participate in the regulation of integrin functions. These results provide insights into the mechanisms by which the central motifs of αIIb and αV tails regulate integrin activation and integrin-mediated cell adhesion

Basal cells in a simple secretory epithelium adhere to the extracellular matrix (ECM), providing contextual cues for ordered repopulation of the luminal cell layer. Early high-grade prostatic intraepithelial neoplasia (HG-PIN) tissue has enlarged nuclei and nucleoli, luminal layer expansion and genomic instability. Additional HG-PIN markers include loss of alpha 6 beta 4 integrin or its ligand laminin-332, and budding of tumor clusters into laminin-511-rich stroma. We modeled the invasive budding phenotype by reducing expression of alpha 6 beta 4 integrin in spheroids formed from two normal human stable isogenic prostate epithelial cell lines (RWPE-1 and PrEC 11220). These normal cells continuously spun in culture, forming multicellular spheroids containing an outer laminin-332 layer, basal cells (expressing alpha 6 beta 4 integrin, high-molecular-weight cytokeratin and p63, also known as TP63) and luminal cells that secrete PSA (also known as KLK3). Basal cells were optimally positioned relative to the laminin-332 layer as determined by spindle orientation. beta 4-integrin-defective spheroids contained a discontinuous laminin-332 layer corresponding to regions of abnormal budding. This 3D model can be readily used to study mechanisms that disrupt laminin-332 continuity, for example, defects in the essential adhesion receptor (beta 4 integrin), laminin-332 or abnormal luminal expansion during HG-PIN progression.

This work was undertaken to characterise the interaction of the leukocyterestricted integrin LFA-1 (CD1la/CD18) with its ligands. LFA-1 function is critical for an immune response and, for example, allows leukocyte binding and transmigration across the endothelium, antigen presentation and cytotoxic T lymphocyte-mediated killing. The ligands for LFA-1 are the Intercellular Adhesion Molecules (ICAMs), with ICAM-1, ICAM-2 and ICAM-3 being the best characterised. The binding sites on ICAM-1 and ICAM-3 for LFA-1 were investigated with the use of antibodies and mutated proteins. The following regions were found to have a role in binding LFA-1: the CFG face of ICAM-3 domain 1; domain 2 of ICAM-1; a residue in domain 1 of ICAM-1 that is mutated at high frequency in African populations and is associated with susceptibility to cerebral malaria. Binding of Mg2+ or Mn 2+ to the extracellular region of LFA-1 and intracellular signalling can both stimulate LFA-1 to adhere to ICAM-1, but by different processes. The former mechanism induces a high affinity form of LFA-1, which was shown to be achieved by an inter-domain movement involving the I domain of the LFA-1 a subunit. This is the first physical evidence for a conformational change occurring in an integrin upon activation. The mechanism by which intracellular signalling activates LFA-1 was demonstrated to involve calpaindependent clustering of LFA-1 in the membrane, thus increasing the avidity of LFA-1 for ICAM-1. Leukocyte Adhesion Deficiency-1 and Glanzmann's Thrombasthenia are genetic disorders in which mutations in the integrin genes result in absence of expression or expression of a non-functional integrin. The defects in function of leukocytes from a patient with clinical features of both disorders were studied. The results suggest that the patient has a novel form of integrin dysfunction in which integrins are expressed at normal levels, can be induced to bind their ligands by mechanisms which increase the affinity of interaction, but cannot be stimulated to bind ligand by intracellular signalling pathways.

Natalizumab ist ein humanisierter monoklonaler Antikörper gegen das Oberflächenadhäsionsmolekül Integrin α-4, der zur Therapie von schweren Verlaufsformen der Multiplen Sklerose (MS) zugelassen ist. Integrin α-4/β-1 wird durch Leukozyten exprimiert und steuert deren Extravasation über die Bindung an VCAM-1 (vascular cell adhesion molecule-1) auf Endothelzellen. Natalizumab wirkt über eine Blockade der Leukozytenadhäsion. In einigen Publikationen konnte darüber hinaus gezeigt werden, dass Integrin α-4 auch auf zerebralen Endothelzellen von Mäusen und Ratten exprimiert wird. In der vorliegenden Arbeit wurde die Expression und Funktion von Integrin α-4 in kultivierten primären humanen zerebralen Endothelzellen untersucht. Die im Rahmen dieser Arbeit an verschiedenen Einzelspenderpräparationen durchgeführten FACS-Analysen zeigten, dass Integrin α-4 in unterschiedlicher Ausprägung auf primären zerebralen Endothelzellisolationen nachzuweisen war. Mit Hilfe immunzytochemischer Färbungen konnte ein spezifisches Verteilungsmuster des Integrin α-4 in Form eines feinen, granulären Musters im Bereich des Zellleibes dokumentiert werden. In Adhäsionsversuchen zeigten Integrin α-4-exprimierende Endothelzellen nach Zugabe von Natalizumab in niedriger Konzentration eine verminderte Fähigkeit zur Haftung an Fibronectin, einem Bindungspartner in der extrazellulären Matrix. In hohen Konzentrationen dominierte im eingesetzten experimentellen System ein unspezifischer Blockadeeffekt, der auch mit Kontrollantikörpern zu beobachten war. In MS-Läsionen findet sich auch die lösliche Form des VCAM-1 (sVCAM-1), die möglicherweise mit endothelialem Integrin α-4 interagiert. Daher wurde mit Hilfe von Western-Blot-Untersuchungen die intrazelluläre Signaltransduktion unter Stimulation mit sVCAM-1 untersucht. Es zeigte sich wie in anderen Endothelarten vorbeschrieben eine Aktivierung des p38-MAP-Kinase-Signalweges. Zusammenfassend wurde demonstriert, dass primäre humane zerebrale Endothelzellen Integrin α-4 exprimieren und dass dieses wahrscheinlich nicht nur für die mechanische Verankerung in der Extrazellulärmatrix eine Bedeutung besitzt, sondern auch als Induktor intrazellulärer Signaltransduktion fungiert, welche die Schrankeneigenschaften zerebraler Endothelzellen beeinflussen könnte. / Expression and function of integrin α-4 in human brain endothelial cells - analysis using theantibody Natalizumab

<p>Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain.</p><p>In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. </p><p>Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent.</p><p>Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.</p>

Integrins are transmembrane glycoproteins primarily mediating interactions of cells with the extracellular matrix. Each receptor is a complex of one α- and one β-subunit with affinity for a diverse set of ligands. A prerequisite for ligand binding, and subsequent events, is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain. In this thesis, the role of a cytoplasmic threonine-cluster, conserved in several β subunits, in β1-integrin activation was investigated. Phosphorylation of these residues is postulated to regulate β2 and β3 integrin affinity for ligands, but it has not been shown so far to occur for β1. Residue T788, but not T789, was established as a site of critical importance for inside-out activation of β1 integrins by mutagenesis to alanine. In contrast to β1-T788A, a phospho-mimicking mutation, β1-T788D, expressed the conformation sensitive 9EG7-epitope and mediated normal cell adhesion. In addition, the T788D mutation did not interfere with binding of the talin head domain, an interaction important for integrin activation. Thus, phosphorylation of T788 in integrin β1 was concluded to be compatible with inside-out receptor activation, in line with β2 and β3 integrin regulation. Focal adhesion kinase (FAK) is activated after integrin ligation and is, together with Src, one of the central players in integrin-mediated events. Phosphatidylinositol 3-kinase (PI3K) is thought to be activated by binding to FAK. However, a novel, major β1-integrin signalling pathway to activate PI3K was identified, which is FAK- and Src-independent. Growth factor induced stimulation of extracellular signal-regulated kinase (Erk) is largely dependent on signals from integrin mediated adhesion to pass checkpoints downstream of Ras. The mechanisms by which β1-integrins mediate Erk-activation were characterized by pin-pointing what phosphorylation sites on the mitogen-activated protein (MAP) kinases and their effector proteins were FAK-dependent. The results indicated that β1 integrins can promote Erk activation by FAK-dependent mechanisms at the levels of both cRaf and Mek, and in addition, a FAK-independent checkpoint at the level of Mek activation.